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A simple purification procedure of buffalo lung cathepsin H, its properties and influence of buffer constituents on the enzyme activity

BACKGROUND: Cathepsin H (E.C.3.4.22.16) belongs to a family of lysosomal cysteine protease which regulates diverse normal biological processes mainly in intracellular proteolysis. METHODS: Purification of cathepsin H from an unstudied system i.e. buffalo lung has been achieved by a simple process de...

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Detalles Bibliográficos
Autores principales: Singh, Shalini, Sharma, Samir, Agarwal, Sudhir K.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Elsevier 2020
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7013249/
https://www.ncbi.nlm.nih.gov/pubmed/32072025
http://dx.doi.org/10.1016/j.bbrep.2020.100739
Descripción
Sumario:BACKGROUND: Cathepsin H (E.C.3.4.22.16) belongs to a family of lysosomal cysteine protease which regulates diverse normal biological processes mainly in intracellular proteolysis. METHODS: Purification of cathepsin H from an unstudied system i.e. buffalo lung has been achieved by a simple process developed after incorporating appropriate alteration in the available methods for isolation of the enzyme from other sources. The use of DEAE-Cellulose and SP-Sephadex C-50 helped in better and simultaneous separation of cathepsin B and H up to homogeneity. RESULTS: The SDS-PAGE result showed buffalo cathepsin H to be a single-chain molecule having MW, NH(2)- and COOH- terminal residues of 25.4 kDa, Lys and Val respectively. The enzyme was a glycoprotein with pI of 6.2; it hydrolyzed Leu-NA (V(max)/K(m) = 301.6) as the most efficient substrate followed by Arg-NA, Arg-Arg-NA and BANA. Buffalo enzyme showed maximum activity at 36 °C, pH 6.75 and at a buffer concentration of 2 × 10(−3) M. CONCLUSION: Catheptic activity was found to be quite stable at least for 20–30 min between pH 4.5–7.0, buffer concentration of 1 × 10(−2) to 4 × 10(−2) M and the temperature resistance up to 36 °C. The effects of various substances present in the buffers routinely used for the assay of catheptic activity revealed that the activity of buffalo lung cathepsin H depends not only qualitatively but also quantitatively on the constituents of assay buffer. GENERAL SIGNIFICANCE: This study seems to provide valuable information regarding the biochemistry of cathepsin H in general as well as influence of buffer constituents on enzyme activity and physiological role in particular.